Clostridium difficile toxins a and/or b antigen and epitope antibody, and pharmaceutical uses thereof

ABSTRACT

It is described a  Clostridium difficile  ( C - difficile ) toxins A and/or B as a target for therapy, including passive immunotherapy, and particularly prevention of  C - difficile  intoxication in human or other animals. It is also described a polypeptide comprising a portion of  C - difficile  toxins A and/or B sequence being an epitope for anti-toxins A and/or B antibody. It is also disclosed a method for generating a neutralizing antibody directed against  C - difficile  toxins A and/or B. It is also provided a novel formulation that combines key toxins A and/or B epitope antibodies, located in three key domains of toxins A and/or B, for neutralisation of the toxins A and/or B, at any stage of toxins A and/or B intoxication related to  C - difficile  infection. The novel formulation of toxins A and/or B epitope antibodies are useful in immunotherapy, for therapeutic and/or prophylactic mediation of  C - difficile  intoxication.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 15/551,848 filed Aug. 17, 2017, which is a national phaseapplication of PCT application No. PCT/CA2016/050170 filed Feb. 19,2016, which claims the benefits of U.S. Provisional Patent ApplicationNo. 62,118,450, filed on Feb. 19, 2015. The content of theseapplications is incorporated herewith by reference.

FIELD OF THE INVENTION

The present invention generally relates to epitopes of toxins A and/or Bproduced by Clostridium difficile and antibodies that specificallybinding to these epitopes. The invention is also related topharmaceutical compositions and vaccines for the prevention or treatmentof Clostridium difficile infection comprising any of the epitopes orantibodies thereof.

BACKGROUND OF THE INVENTION

Clostridium difficile is a Gram-positive, anaerobic, endospore-forminggastrointestinal pathogen responsible for C-difficile-associated disease(CDAD) in humans and animals with symptoms ranging in severity from mildcases of antibiotic-associated diarrhea to fatal pseudomembranouscolitis (Rupnik et al, 2009; Leffler and Lamont, 2009; Songer, 2004;Kelly et al, 1994). Each year in North America, 1-3% of hospitalizedpatients receiving antibiotics become infected with C-difficile, leadingto thousands of deaths and over $1 billion in associated costs to thehealth-care system (Wilkins and Lyerly, 2003; Kyne et al, 2002; Kelly etal, 1994). C-difficile produces two primary virulence factors, toxin A(TcdA) and toxin B (TcdB), which are large (308 kDa and 269 kDa,respectively), single-subunit exotoxins composed of a catalytic, atranslocation and a cell-receptor binding domain (RBD) (Jank andAktories, 2008; Jank et al, 2007). Recently it was suggested TcdB issolely responsible for C-difficile virulence (Lyras et al, 2009),although earlier studies have shown both anti-TcdA and anti-TcdBmonoclonal antibodies (mAbs) were required for full protection ofhamsters from CDAD (Babcock et al, 2006; Kink and Williams, 1998) andanti-TcdA mAbs were required for protection in mice (Corthier et al,1991).

The current approach for treating most CDAD infections involvesadministration of antibiotics, most commonly metronidazole or vancomycin(Leffler and Lamont, 2009). Antibiotic treatment places selectionpressure on the organism, can lead to antibiotic resistance, andsuppresses or eliminates beneficial commensal microbes. However, thereare several other emerging challenges warranting the development ofnovel therapeutics. First, there is no acute CDAD treatment targetingTcdA and/or B. These toxins are responsible for loss of epithelialbarrier function in the colon by disrupting tight junctions andincreasing membrane permeability, causing diarrhea and promoting severeinflammation (Rupnik et al, 2009; Jank and Aktories, 2008). Second,hypervirulent strains of C-difficile, such as the NAP1/027 isolate,over-express TcdA and TcdB (Warny et al, 2005) and have been associatedwith increased mortality rates and disease severity (O′Connor et al,2009; Pepin et al, 2005). Third, an estimated 20-25% of patientssuffering from CDAD experience symptomatic relapse after the initialinfection is cleared, with 45% of these patients prone to subsequentrelapses (Johnson, 2009). Taken together, there is a need fornon-antibiotic based reagents targeting and inhibiting TcdA and TcdB forCDAD therapy. Individuals who are asymptomatic C-difficile carriers andpatients who experience mild cases of CDAD tend to possess highanti-toxin A titers (Kyne et al, 2001; Kyne et al, 2000; Warny et al,1994; Viscidi et al, 1983). Conversely, patients susceptible torelapsing C-difficile infection have low anti-TcdA immunoglobulintiters, specifically IgM, lgG2 and lgG3 isotypes (Katchar et al, 2007;Kyne et al, 2001). TcdA-neutralizing secretory IgA antibodies are alsothought to play a role in regulating CDAD severity (Johal et al 2004;Kelly et al 1992). Therefore, the introduction of anti-toxin antibodiesto patients suffering from severe C-difficile infection may be atherapeutically useful approach.

A limited number of animal and human studies have illustrated theeffectiveness of anti-toxin Abs for treatment of CDAD. Babcock et al(2006) intravenously administered anti-TcdA and anti-TcdB mAbs tohamsters and found a significant reduction in hamster mortality inprophylactic, primary disease and relapse models when both anti-toxinmAbs were administered. A recently completed clinical trial involvingthese two humanized mAbs appears promising (Lowy et al, 2010). Inanother study, intravenous administration of anti-TcdA mAbs raisedagainst the RBD followed by oral challenge with C-difficile resulted inprotection of mice (Corthier et al, 1991). Elsewhere, a toxoid vaccinegiven by the intraperitoneal route to hamsters conferred protectionagainst oral C-difficile challenge (Giannasca et al, 1999) and micevaccinated with DNA encoding the TcdA RBD resulted in full protectionfrom oral TcdA challenge (Gardiner et al, 2009). In humans, a number ofuncontrolled studies have reported intravenous immunoglobulin (IVIG)therapy to be successful for the treatment of severe CDAD (Juang et al,2007; Hassoun and Ibrahim, 2007; McPherson et al, 2006; Wilcox, 2004;Salcedo et al, 1997; Leung et al, 1991). IVIG involves administration ofhigh concentrations (150-400 mg/kg) of human immunoglobulins fromhealthy donors which are thought to contain neutralizing anti-toxinantibodies as an estimated 60% of healthy adults have detectable TcdA-and TcdB-specific serum IgG antibodies (Viscidi et al, 983). Given thatC-difficile toxins rely on attachment to epithelial cells for entry(Jank and Aktories, 2008; Jank et al, 2007), neutralizing the toxinswithin the lower gastrointestinal tract with antibodies may block thefirst step in CDAD pathogenesis. In animals, orally administered bovineimmunoglobulin concentrate (BIC) containing TcdA and TcdB neutralizingIgGs were able to prevent hamster mortality when used as a propholyactic(Lyerly et al, 1991) and protected rats from the enterotoxic effects ofTcdA in vivo (Kelly et al, 1996). Chicken IgY antibodies specific fortoxin RBDs were shown to reduce hamster mortality when administeredorally to infected animals (Kink and Williams, 1998). In humans, therehave been limited reports on CDAD therapy with orally delivered Abs.Tjellstrom et al (1993) reported the successful treatment of a 3½ yearold boy suffering from severe CDAD with IgA antibody orally. Warny et al(1999) and Kelly et al (1997) examined the passage of anti-toxin bovineIgG through the human gastrointestinal tract and found a significantreduction in IgG activity, likely due to proteolytic degradation withinthe upper gastrointestinal tract. The limited success of both oral andsystemic anti-toxin immunotherapy in clinical settings has likely beenhampered by the high immunoglobulin dose requirements (150-400 mg/kg),the associated costs of these doses, and a lack of published clinicaldata showing the effectiveness of these treatments.

Despite such advances, there remains a need in the art for a safe andeffective therapeutic for treating C-difficile-associated disease aswell as for sensitive and effective reagents for the detection of toxinsA and B, the factors responsible for C-difficile-associated disease.

SUMMARY OF THE INVENTION

The aforesaid and other objectives of the present invention are realizedby generally providing specific Clostridium difficile (C-difficile)toxins A and/or B as a target for therapy, including passiveimmunotherapy, and particularly prevention of toxins A and/or Bintoxication in human or other animals.

One aspect of the present invention is to provide an isolatedpolypeptide comprising a portion of Clostridium difficile toxins Aand/or B sequence, the portion of toxins A and/or B sequence being anepitope for anti-toxins A and/or B antibody, the portion comprising asequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,or any combination thereof.

Preferably, the portion of toxins A and/or B sequence may comprise acombination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4.

The aforesaid isolated peptides may be used for immunizing an animalagainst Clostridium difficile infection.

Another aspect of the present invention is to provide a pharmaceuticalcomposition for generating a neutralizing antibody directed againstClostridium difficile toxins A and/or B and comprising at least twodifferent polypeptides, each polypeptide comprising a portion ofClostridium difficile toxins A and/or B sequence, the portion of toxinsA and/or B sequence being an epitope for anti-toxins A and/or Bantibody, and the portion comprising a sequence being SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

Preferably, the pharmaceutical composition may comprise four differentpolypeptides, wherein the portion of toxins A and/or B sequencecomprises a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 andSEQ ID NO: 4.

The aforesaid pharmaceutical compositions may further comprise apharmaceutically acceptable carrier.

The pharmaceutical compositions may be used for immunizing an animalagainst Clostridium difficile infection.

Another aspect of the present invention is to provide a vaccinecomposition for prevention or treatment of Clostridium difficileinfection comprising at least one polypeptide which comprises a portionof Clostridium difficile toxins A and/or B sequence, the portion oftoxins A and/or B sequence being an epitope for anti-toxins A and/or Bantibody, and the portion comprising a sequence being SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

Preferably, the aforesaid vaccine composition may comprise the portionof toxins A and/or B sequence comprising a combination of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

The aforesaid vaccine compositions may further comprise apharmaceutically acceptable adjuvant.

Another aspect of the present invention is to provide a nucleic acidvaccine or DNA vaccine composition for prevention or treatment ofClostridium difficile infection comprising nucleic acids encoding atleast one polypeptide which comprises a portion of Clostridium difficiletoxins A and/or B sequence, the portion of toxins A and/or B sequencebeing an epitope for anti-toxins A and/or B antibody; the portioncomprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, or any combination thereof.

Preferably, the aforesaid nucleic acid vaccine composition may comprisethe portion of toxins A and/or B sequence comprising a combination ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

The aforesaid nucleic acid vaccine compositions may further comprise apharmaceutically acceptable adjuvant.

Another aspect of the present invention is to provide a method ofgenerating a neutralizing antibody directed against Clostridiumdifficile toxins A and/or B. The method comprises a first step ofadministrating to a host an isolated polypeptide comprising a portion ofthe Clostridium difficile toxins A and/or B sequence, the portioncomprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, or any combination thereof, for generating antibodies inthe host. The method also comprises a second step of obtaining theantibodies from the host. The host may be a mammal or a bird, includingbird eggs, such as but not limited to chicken eggs.

Preferably, in the first step of the method, the portion of theClostridium difficile toxins A and/or B sequence comprises a combinationof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

Another aspect of the present invention is to provide a purifiedantibody adapted for binding to a toxins A and/or B peptide, the peptidecomprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, or any combination thereof.

The aforesaid antibody may also be an active fragment thereof, achimeric antibody, a veneered antibody, a humanized antibody or a singlechain recombinant antibody based thereon as well as a bird polyclonalantibody or a bird humanized recombinant antibody.

The aforesaid antibody may be used for detecting pure toxins A and/or Bor a presence of toxins A and/or B produced by Clostridium difficile incell culture.

Another aspect of the present invention is to provide an antibodycomposition for prevention or treatment of Clostridium difficileinfection, comprising different antibodies adapted for binding to atleast two epitopes of toxins A and/or B, the at least two epitopescomprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, or any combination thereof.

Preferably, the aforesaid antibody composition may comprises differentantibodies adapted for binding to four different epitopes, the epitopescomprising a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 andSEQ ID NO: 4.

Another aspect of the present invention is to provide a pharmaceuticalcomposition for the prevention or the treatment of Clostridium difficiletoxins A and/or B intoxication, the composition comprising at least oneantibody adapted for binding to at least one epitope of toxins A and/orB, the at least one epitope comprising a sequence being SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

Preferably, the aforesaid pharmaceutical composition may comprises atleast one epitope comprising a combination of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO: 4.

The aforesaid pharmaceutical compositions may further comprise apharmaceutically acceptable carrier.

The aforesaid pharmaceutical compositions may be used for making amedicament for preventing or treating Clostridium difficile infection.

The aforesaid pharmaceutical compositions may also be used for thecapture and neutralisation of Clostridium difficile toxins A and/or B,allowing a passive immunotherapy of mammals, such as humans.

The features of the present invention which are believed to be novel areset forth with particularity in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the inventionwill become more readily apparent from the following description,reference being made to the accompanying drawings in which:

FIGS. 1A and B are schematic representation of the targeted portion oftoxins A and/or B epitope antibodies.

FIGS. 2A and B illustrates the targeted sequence portion of toxins Aand/or B epitope antibodies.

FIG. 3 is a photograph of a western blot showing recognition of toxins Aand/or B by toxins A and/or B epitope antibody IBSCD1, according to apreferred embodiment of the present invention.

FIG. 4 is a photograph of a western blot showing recognition of toxins Aand/or B by toxins A and/or B epitope antibody IBSCD2, according to apreferred embodiment of the present invention.

FIG. 5 is a photograph of a western blot showing recognition of toxins Aand/or B by toxins A and/or B epitope antibody IBSCD3, according to apreferred embodiment of the present invention.

FIG. 6 is a photograph of a western blot showing recognition of toxins Aand/or B by toxins A and/or B epitope antibody IBSCD4, according to apreferred embodiment of the present invention.

FIGS. 7A-D is a set of microscope photographs showing the neutralisationeffect on the cell rounding and dead of Caco-2 intestinal cell line bytoxins A and/or B epitope antibodies formulation in presence of thesupernatant of C-difficile NAP1/027 hypervirulent strain, according to apreferred embodiment of the present invention.

FIGS. 8A-F is a set of microscope photographs showing the neutralisationeffect on the cell rounding and dead of Caco-2 intestinal cell line bytoxins A and/or B epitope antibodies formulation in presence of thesupernatant of C-difficile NAP1/027 hypervirulent strain and purifiedtoxin A and toxin B, according to a preferred embodiment of the presentinvention.

FIGS. 9A-F is a set of epifluorescence microscope photographs showingimprovement of Caco-2 cell viability by the blocking antibodies againstC-difficile toxin A and/or B, according to a preferred embodiment of thepresent invention.

FIGS. 10A-C is a set of graphs showing the protection of the Caco-2monolayer integrity by the blocking antibodies against C-difficile toxinA and/or B, according to a preferred embodiment of the presentinvention.

FIGS. 11A and B is a set of graphs showing the reduction of Clostridiumdifficile infection in vivo by the blocking antibodies againstC-difficile toxin A and/or B, according to a preferred embodiment of thepresent invention.

FIGS. 12A-C is a set of microscope photographs showing the reduction ofmucosal damage in murine colon by the blocking antibodies againstC-difficile toxin A and/or B, according to a preferred embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A novel composition of anti-toxins A and/or B antibodies and toxins Aand/or B epitopes will be described hereinafter. Although the inventionis described in terms of specific illustrative embodiment(s), it is tobe understood that the embodiment(s) described herein are by way ofexample only and that the scope of the invention is not intended to belimited thereby.

A broad aspect, the present invention provides a method that uses acombination of toxins A and/or B epitope antibodies, including but notlimited to bird's epitope antibodies, for the capture and neutralisationof C-difficile toxins A and/or B, which permit a passive immunotherapyof humans or others mammals. The method of neutralisation aims threedifferent key portions of toxins A and/or B among the four domains ofC-difficile toxins, which are the combined repetitive oligopeptidesc-terminal domain (CROPs) 10, the delivery pore forming domain 20, theauto-protease domain 30, and the N-terminal glucosyltransferase domain40, as illustrated in FIGS. 1 and 2. These different portions of toxinsA and/or B includes four different epitopes adapted for generatingtoxins A and/or B epitope antibodies in mammals or birds. Furthermore,FIG. 2 shows the location of the four epitopes within the toxins A and Bamino acid sequences as part of the present invention.

According to a first embodiment of the present invention, it is providedan isolated peptide having the amino acid sequence: DSKKYYFNTNTAEAA (SEQID NO: 1). This particular peptide is a toxins A and/or B epitopepeptide encompassing amino acids 2084-2098 of toxins A and/or B, whichhas been identified as a shared toxins A and/or B epitope using thechicken polyclonal antibodies IBSCD1 (see FIG. 3).

According to a second embodiment of the present invention, it isprovided an isolated peptide having the amino acid sequence:ANQYEVRINSEGR (SEQ ID NO: 2). This particular peptide is a toxins Aand/or B epitope peptide encompassing amino acids 739-751 of toxins Aand/or B, which has been identified as a shared toxins A and/or Bepitope using the chicken polyclonal antibodies IBSCD2 (see FIG. 4).

According to a third embodiment of the present invention, it is providedan isolated peptide having the amino acid sequence GHGKDEFNTDIFAG (SEQID NO: 3). This particular peptide is a toxins A and/or B epitopepeptide encompassing amino acids 652-665 of toxins A and/or B, which hasbeen identified as a shared toxins A and/or B epitope using the chickenpolyclonal antibodies IBSCD3 (see FIG. 5).

According to a forth embodiment of the present invention, it is providedan isolated peptide having the amino acid sequence DEYNKLTTNNNENKYL (SEQID NO: 4). This particular peptide is a toxins A and/or B epitopepeptide encompassing amino acids 31-46 of toxins A and/or B, which hasbeen identified as a shared toxins A and/or B epitope using the chickenpolyclonal antibodies IBSCD4 (see FIG. 6).

In accordance with another embodiment of the present invention, theisolated peptides, including combinations of one or more thereof, areadapted for generating antibodies which recognize toxins A and/or B andhave a neutralising therapeutic or prophylactic activity in immunizinganimals, particularly mammals, most particularly humans, who have aC-difficile intoxication.

In accordance with a preferred embodiment of the present invention, itis provided a combination of polypeptides, which comprises all theafore-mentioned amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 and SEQ ID NO: 4, or any combination thereof.

The afore-mentioned polypeptides may include an immunogenic peptide,particularly comprising amino acid sequence of any of SEQ ID NOS: 1-4,or an immunogenic fragment thereof. These polypeptides may also includeimmunogenic receptor of peptides, wherein such polypeptides comprise acombination of at least one immunogenic receptor peptide comprisingamino acid sequence of any of SEQ ID NOS: 1-4, or immunogenic peptidefragment thereof.

In accordance with another embodiment of the present invention, it isprovided a method for immunizing an animals, particularly mammals orbirds comprising administering a toxins A and/or B epitope peptide or animmunogenic fragment thereof, whereby the animal produces antibodiesthat are immunoreactive with the epitope peptide exposed on partial orfull length toxins A and/or B produced by C-difficile bacteria. Themethod for immunizing mammals or birds may comprise administering atoxins A and/or B peptide comprising amino acid sequence of any of SEQID NOS: 1-4 or an immunogenic fragment thereof, whereby the animalproduces antibodies that are immunoreactive to full length toxins Aand/or B produced by C-difficile bacteria. According to a preferredembodiment, the aforesaid method comprises the use of the four differenttoxins A and/or B peptides with the amino acid sequences of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

In accordance with another embodiment of the present invention, it isprovided a pharmaceutical composition for generating a neutralizingantibody directed against Clostridium difficile toxins A and/or B, thepharmaceutical composition comprising a toxins A and/or B peptide,particularly with amino acid sequence of any of SEQ ID NOS: 1-4, and apharmaceutically acceptable carrier. According to a preferredembodiment, the pharmaceutical composition comprises the four differenttoxins A and/or B peptides with the amino acid sequences of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceuticallyacceptable carrier.

In accordance with another embodiment of the present invention, it isprovided a vaccines or immunogenic compositions that comprise one ormore toxins A and/or B peptide, particularly with amino acid sequence ofany of SEQ ID NOS: 1-4, and a pharmaceutically acceptable adjuvant.According to a preferred embodiment, the vaccine composition comprisesthe four different toxins A and/or B peptides with the amino acidsequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4,and a pharmaceutically acceptable adjuvant.

The vaccine may also be used for treatment of a subject, such as amammal, particularly a human subject, suffering from a C-difficileintoxication. Such vaccine may comprise an immunogenic amount of one ormore toxins A and/or B peptides, particularly with amino acid sequenceof any of SEQ ID NOS: 1-4 or immunogenic fragment thereof, and apharmaceutically acceptable adjuvant. The aforesaid toxins A and/or Bpeptides may be conjugated to a carrier.

In accordance with another embodiment of the present invention, it isprovided a nucleic acid vaccines or DNA vaccines comprising nucleicacids encoding immunogenic toxins A and/or B peptides, particularly withamino acid sequence of any of SEQ ID NOS: 1-4, and a pharmaceuticallyacceptable adjuvant. According to a preferred embodiment, the nucleiacid vaccine composition comprises the nucleic acids encoding the fourdifferent toxins A and/or B peptides with the amino acid sequences ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and apharmaceutically acceptable adjuvant. The afore-mentioned nucleic acidvaccine may further comprises at least one other polypeptide,particularly an immunomodulatory molecule peptide derived fromC-difficile.

In accordance with another embodiment of the present invention, it isprovided a method for diagnosis of Clostridium difficile infectioncomprising the steps of contacting a biological sample of a subject withat least one peptide fragment of toxins A and/or B and detectingantigen-antibody complex formation. Such at least one peptide fragmentmay comprises an epitope for anti-toxins A and/or B antibody with anamino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, or any combination thereof.

In accordance with another embodiment of the present invention, it isprovided a kit for the diagnosis or detection of Clostridium difficileinfection, the kit comprising at least one peptide fragment of toxins Aand/or B and directions for diagnosing or detecting anti-toxin A and/orB antibody. Such at least one peptide fragment may comprise an epitopefor anti-toxin A and/or B antibody with an amino acid sequence of any ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or anycombination thereof.

As previously mentioned, toxins A and/or B polypeptides with the aminoacid sequence of any of SEQ ID NOS: 1-4 are adapted for generatingtoxins A and/or B epitope antibodies in mammals or birds. Suchantibodies recognize toxins A and/or B and have neutralising effect onthe activity of these toxins, so that the antibodies may be used for atherapeutic or prophylactic treatment against C-difficile intoxicationin humans and animals.

In accordance with another embodiment of the present invention, it isprovided a method of generating a neutralizing antibody directed againstClostridium difficile toxins A and/or B. The method comprises a firststep of administrating to a host an isolated polypeptide comprising aportion of the Clostridium difficile toxins A and/or B sequence. Theportion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, or any combination thereof, for generatingantibodies in the host. The method comprises a second step of obtainingthe antibodies from the host. The host may be a mammal or a bird,including the bird eggs, such as but not limited to chicken eggs.

The aforesaid method may also comprise a first step, wherein the portionof the Clostridium difficile toxins A and/or B sequence comprises asequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4.

Referring to FIG. 3, an antibody, called IBSCD1, is adapted for bindingto a key toxins A and/or B epitope, encompassing amino acids 2084-2098of toxin A and/or B located in combined repetitive oligopeptidesc-terminal domain (CROPs) 10 of toxins A and/or B. This domain plays arole in the binding to human or animal cells, as illustrated in FIG. 1,step 1.

Referring to FIGS. 4 and 5, two antibodies, called IBSCD2 and IBSCD3,are adapted for binding to toxins A and/or B epitopes, encompassingrespectively amino acids 739-751 and 652-665 of toxins A and/or Blocated in the delivery pore forming domain 20. This domain plays a rolein the endosome pore formation of the infected cell, as illustrated inFIG. 1, step 3.

Referring to FIG. 6, another antibody, called IBSCD4, bind the toxins Aand/or B epitope encompassing amino acids 31-46 of toxins A and/or Blocated in the glucosyltransferase domain 40. This domain plays a rolein the inactivation of small GTPases in the affected cells, asillustrated in FIG. 1, step 4.

In accordance with a preferred embodiment of the present invention, itis provided a novel formulation that combines key toxins A and/or Bepitope antibodies, located in three key domains of toxins A and/or B,for neutralisation of the toxins A and/or B, at any stage of toxins Aand/or B intoxication related to C-difficile infection. Therefore, thenovel formulation of toxins A and/or B epitope antibodies may be used inimmunotherapy, for therapeutic and/or prophylactic mediation ofC-difficile intoxication.

In accordance with another embodiment, it is provided a purifiedantibody to a toxin A and/or B peptide comprising the amino acidsequence of any of SEQ ID NOS: 1-4. The above-described antibodies mayspecifically detect pure toxins A and/or B or the presence of thesetoxins produced by C-difficile in culture.

The antibody may be selected from antibodies IBSCD1, IBSCD2, IBSCD3,IBSCD4 or active fragments thereof. The antibody may include bothpolyclonal and monoclonal antibodies prepared by known genetictechniques, as well as bi-specific antibodies, and antibodies includingother functionalities suiting them for diagnostic or therapeutic use.The antibody may consist of bird polyclonal or bird humanizedrecombinant antibodies. Furthermore, the antibody may include, but notlimited to, naturally raised and recombinant prepared antibodies orfragments thereof, including single chain variants and Fv. Theantibodies may also include chimereic antibodies, veneered antibodies,humanized antibodies, chicken polyclonal antibodies, chicken recombinanthumanized antibodies, domain antibodies, calemized antibodies and singlechain recombinant antibodies. Such antibodies can be used for passiveimmunization to reduce C-difficile intoxication, particularly in humans.

In accordance with another embodiment of the present invention, it isprovided a pharmaceutical composition for preventing or treatingC-difficile toxins A and/or B intoxication. The pharmaceuticalcomposition may comprise at least one antibody adapted for binding to atleast one epitope of toxins A and/or B, and a pharmaceuticallyacceptable carrier. Such epitope may comprise a amino acid sequence ofany of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or anycombination thereof. According to a preferred embodiment, thepharmaceutical composition comprises the antibodies adapted for bindingto the four different toxins A and/or B peptides with the amino acidsequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4,and a pharmaceutically acceptable carrier.

In accordance with a another embodiment of the present invention, it isprovided therapeutic methods based upon the activity of an antibody, oractive fragments thereof, adapted for binding to a toxins A and/or Bpeptide comprising amino acid sequence of any of SEQ ID NOS: 1-4. Inparticular, the method may comprise antibodies, or active fragmentsthereof, and chimeric or synthetic antibodies derived therefrom, and canbe prepared in pharmaceutical compositions, including a suitablevehicle, carrier or diluent, for administration in instances whereintherapy is appropriate, such as to treat C-difficile infection. Suchmethods may include oral formulations of avian of mammal anti-toxins Aand/or B for prevention of toxins A and/or B intoxication. Such methodsmay also include modulating the half-life of the binding members,antibodies or fragments by methods known in the art such as pegylation.Such methods may further comprise additional antibodies or therapeuticagents.

In accordance with a another embodiment of the present invention, it isprovided a method for diagnosis of Clostridium difficile infectioncomprising the steps of contacting a biological sample of a subject withat least one anti-toxins A and/or B antibody adapted for binding to atleast one epitope of toxins A and/or B, and detecting antigen-antibodycomplex formation. Such at least one epitope may comprise an amino acidsequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, or any combination thereof.

In accordance with a another embodiment of the present invention, it isprovided a kit for the diagnosis or detection of Clostridium difficileinfection, the kit comprising at least one anti-toxins A and/or Bantibody adapted for binding to at least one epitope of toxins A and/orB, and directions for diagnosing or detecting anti-toxin A and/or Bantibody. Such at least one epitope may an amino acid sequence of any ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or anycombination thereof.

EXAMPLES

FIGS. 7A-D the neutralisation effect on the cell rounding and dead ofCaco-2 intestinal cell line by toxins A and/or B epitope antibodiesformulation in presence of the supernatant of C-difficile NAP1/027hypervirulent strain. FIG. 7A shows intact monolayer of Caco-2 cellsafter incubation with 25 μ1 of water (negative control). FIG. 7B showsdisturbed and destroyed monolayer of Caco-2 cells, with a lot of cellrounding and dead cells after incubation with 25 μl of undilutedNAP1/027 strain C-difficile toxin A/B supernatant. FIG. 7C showsdisturbed monolayer of Caco-2 cells, with cell rounding (arrows) anddead cells after incubation with 25 μl of 1:1000 dilution of NAP1/027strain C-difficile toxin A/B supernatant. FIG. 7D shows preservedmonolayer of Caco-2 cells after incubation with 25 μl of 1:1000 dilutionof NAP1/027 strain C-difficile toxin A/B supernatant in the presence ofa formulation containing 63 μg/ml of IBSCD1, IBSCD2, IBSCD3 and IBSCD4toxins A/B epitope antibodies.

FIGS. 8A-F show the neutralisation effect on the cell rounding and deadof Caco-2 intestinal cell line by adding toxins A and/or B epitopeantibodies formulation when in presence of the supernatant ofC-difficile NAP1/027 hypervirulent strain and purified toxin A and toxinB. FIG. 8A shows complete rounding of cells, lost of adhesion and cellsdeath after incubation with 400 ng/mL of purified toxin A. FIG. 8B showsreduction of cytotoxic effect, with decrease in rounding and cell death,after incubation with 400 ng/mL toxin A, preincubated with 63 μg/mL ofIBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies.FIG. 8C shows disturbed monolayer of Caco-2 cells, with cell roundingand dead cells and without dome formation, after incubation with 25 μlof 1:100 dilution of NAP1/027 strain C-difficile toxin A/B supernatant.FIG. 8D shows preserved monolayer of Caco-2 cells after incubation with25 μl of 1:100 dilution of NAP1/027 strain C-difficile toxin A and/or Bsupernatant in the presence of a formulation containing 125 μg/ml ofIBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies.FIG. 8E shows complete rounding of cells, lost of adhesion and celldeath after incubation with 10 ng/mL of purified toxin B. FIG. 8F showsreduction of cytotoxic effect, with decrease in rounding and cell deathafter incubation with 40 ng/mL toxin B, preincubated with 125 μg/mL ofIBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies.

FIGS. 9A-F show cell death induced by C. difficile toxin A and B inCaco-2 cells and the improvement of cell viability by the blockingantibodies. Caco-2 cells were seeded in chamber slides in 200 μL ofculture medium and incubated overnight at 37° C. in CO2 incubator. Thefollowing day, cell death was induced with toxin A and B and cells wereincubated for 48 h. Cells were washed with PBS and incubated withAnnexin V and PI (1:10) for 15 min. Coverslips were mounted withmounting medium and DAPI. Images were taken by epifluorescencemicroscopy. FIG. 9A shows little or no cell death in cells treated withwater. FIG. 9B shows little or no cell death due to a normal process ofcell death in the presence of 125 μg/mL of IBSCD1, IBSCD2, IBSCD3 andIBSCD4 Toxins A and/or B epitope antibodies. FIG. 9C shows increased innumber of green (or white), Annexin V labelled cells representingapoptotic cells, in the presence of 400 ng/mL toxin A. FIG. 9D shows adecrease in cell death with 400 ng/mL toxin A preincubated with 125μg/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitopeantibodies. FIG. 9E shows an increase in cell death with 40 ng/mL toxinB. FIG. 9F shows a decrease in cell death with 40 ng/mL toxin Bpreincubated with 125 μg/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxinsA/B epitope antibodies. C. difficile toxins A and B induced programmedcell death and even necrosis (represented in white), while preincubationof toxins with IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A/B epitopeantibodies deceased number of apoptotic cells in all conditions. Bleu,DAPI labelled cells represent live cells.

FIGS. 10A-C show the protection of the integrity of Caco-2 monolayer bythe blocking antibodies against C. difficile toxin A and B. FIG. 10Ashows 7-10 days post-confluent Caco-2 monolayer that was grown on porous4 μm inserts. Only polarized monolayers of cell were used. At 6 h, thetransepithelial electric resistance (TEER) of the Caco-2 monolayerrapidly decreased down to 67% when cells were stimulated with 400 ng/mltoxin A. Cells treated with toxin A preincubated with blockingantibodies IBSCD1, IBSCD2, IBSCD3 and IBSCD4 increased TEER up to 86%compared to unstimulated cells, which were reported to one. FIG. 10Bshows the rapid decrease of the transepithelial electric resistance(TEER) of the Caco-2 monolayer down to 77% when cells were stimulatedwith 40 ng/ml toxin B compared to unstimulated cells. Use of 125 μg/m ofblocking antibodies restored epithelial integrity up to 93%. FIG. 10Cshows that when cells were stimulates with supernatant of C. difficileNAP1/027 strain (1:10), TEER decreased to 61% compared to unstimulatedafter 6 h. Preincubation of 125 μg/ml IBSCD1, IBSCD2, IBSCD3 and IBSCD4toxins A and/or B epitope antibodies with supernatant restoredepithelial integrity up to 84%. Results represent the mean SEM of threeindependent experiments performed in duplicate. Statistical analysis wasdone with 2 way ANOVA.

FIGS. 11A-B show the reduction of Clostridium difficile infection invivo by the blocking antibodies against C-difficile toxin A and/or B.6-8 weeks old female mice were housed in groups of 4 in sterile cagesequipped with HEPA filters and containing sterile bedding. They hadaccess to sterile food and water ad libitum. Mice are intrinsicallyresistant to CDI, so they were be pre-conditioned for three days with250 mg/L clindamycin and 400 mg/L streptomycin in the drinking water,followed by an intraperitoneal (i.p.) injection of 1 mgclindamycin/mouse to disrupt their intestinal microbiota and make themsusceptible to CDI. Twenty-four hours later, mice received by gavage10e+5 spores of the epidemic CD strain R20291 to initiate the infection.Mice were gaved twice a day with 1mg of a preparation of IBSCD1, IBSCD2,IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies in 0.1 Mcarbonate buffer (pH 9.2) to neutralize the gastric acidity for 5 days.Control groups consist of uninfected mice, and infected but untreatedmice. FIG. 11A shows the results from mice that were observed for atotal of 7-10 days and clinical symptoms were monitored daily (diarrhea,weight loss, lethargy, etc.). FIG. 11B shows results from fresh fecalsamples that were collected daily and homogenized in pre-reduced PBS,and CD were enumerated on agar plates. Clinical score end bacterialcounts were the higher in untreated mice in comparison with uninfectedmice. Mice receiving IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or Bepitope antibodies had lower CDI score then untreated mice and lowerbacterial counts.

FIGS. 12A-C show the reduction of mucosal damage in murine colon by theblocking antibodies against C-difficile toxin A and/or B. 6-8 weeks oldfemale mice were housed in groups of 4 in sterile cages equipped withHEPA filters and containing sterile bedding. They had access to sterilefood and water ad libitum. Mice are intrinsically resistant to CDI, sothey were be pre-conditioned for three days with 250 mg/L clindamycinand 400 mg/L streptomycin in the drinking water, followed by anintraperitoneal (i.p.) injection of 1 mg clindamycin/mouse to disrupttheir intestinal microbiota and make them susceptible to CDI.Twenty-four hours later, mice received by gavage 10e+5 spores of theepidemic CD strain R20291 to initiate the infection. Mice were gavedtwice a day with 1 mg of a preparation of IBSCD1, IBSCD2, IBSCD3 andIBSCD4 toxins A and/or B epitope antibodies in 0.1 M carbonate buffer(pH 9.2) to neutralize the gastric acidity for 5 days. Control groupsconsist of uninfected mice, and infected but untreated mice. At day ten,colons were extracted and embedded in paraffin. Hematoxylin & Eosin(H&E) staining was performed. FIG. 12A shows normal H&E staining ofuninfected mice. FIG. 12B shows H&E staining of infected but untreatedmice, which presented signs of mild inflammation. FIG. 12C shows H&Estaining of infected mice treated with IBSCD1, IBSCD2, IBSCD3 and IBSCD4toxins A and/or B epitope antibodies, which presented less severemucosal damage then untreated mice. Black arrows indicate thickness ofsubmucosal layer, immune cell infiltration and lost of epithelial layer.

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While illustrative and presently preferred embodiment(s) of theinvention have been described in detail hereinabove, it is to beunderstood that the inventive concepts may be otherwise variouslyembodied and employed and that the appended claims are intended to beconstrued to include such variations except insofar as limited by theprior art.

What is claimed is: 1) An antibody that recognizes Clostridium difficiletoxins A and B, wherein said antibody specifically binds to the sequenceset forth in SEQ ID NO: 1 or SEQ ID NO:2. 2) The antibody of claim 1,wherein said antibody specifically binds to the sequence set forth inSEQ ID NO:
 1. 3) The antibody of claim 1, wherein said antibodyspecifically binds to the sequence set forth in SEQ ID NO:
 2. 4) Theantibody of claim 1, wherein said antibody is a polyclonal antibody. 5)The antibody of claim 1, wherein said antibody is a bird antibody. 6)The antibody of claim 5, wherein said bird antibody is a chickenantibody. 7) The antibody of claim 1, wherein said antibody is in acomposition that further comprises a carrier. 8) The antibody of claim7, wherein said composition is a pharmaceutical composition and saidcarrier is a pharmaceutically acceptable carrier. 9) A method forimmunizing a subject against Clostridium difficile infection or treatingClostridium difficile infection in a subject comprising administratingan effective amount of the antibody of claim 1 to a subject in needthereof. 10) A method for immunizing a subject against Clostridiumdifficile infection or treating Clostridium difficile infection in asubject comprising administrating an effective amount of the antibody ofclaim 4 to a subject in need thereof. 11) A method for immunizing asubject against Clostridium difficile infection or treating Clostridiumdifficile infection in a subject comprising administrating an effectiveamount of the antibody of claim 6 to a subject in need thereof. 12) Themethod of claim 9, wherein said subject is a human subject. 13) Themethod of claim 10, wherein said subject is a human subject. 14) Themethod of claim 11, wherein said subject is a human subject.